Generation of human colon organoids from healthy and inflammatory bowel disease mucosa

Ulcerative colitis and Crohn’s disease are chronic inflammatory bowel diseases (IBD) of unknown cause characterized by a relapsing-remitting behavior. Growing evidence supports the idea that the epithelial barrier plays a central role in the pathogenesis of IBD as well as in its evolution over time, thus representing a potential target for novel therapeutic options. In the last decade, the introduction of 3D epithelial cultures from ex vivo-expanded intestinal adult stem cells (ASCs) has impacted our ability to study the function of the epithelium in several gastrointestinal disorders, including IBD. Here, we describe in detail a reproducible protocol to generate Matrigel-embedded epithelial organoids from ASCs of non-IBD and IBD donors using small colonic biopsies, including steps for its optimization. A slightly modified version of this protocol is also provided in case surgical samples are used. With this method, epithelial organoids can be expanded over several passages, thereby generating a large quantity of viable cells that can be used in multiple downstream analyses including genetic, transcriptional, proteomic and/or functional studies. In addition, 3D cultures generated using our protocol are suitable for the establishment of 2D cultures, which can model relevant cell-to-cell interactions that occur in IBD mucosa.

To prepare aliquots from a new Matrigel batch, thaw the bottle overnight on ice in a cold room, and prepare small aliquots using a cold 5 mL pipette and 1.5 mL Eppendorf tubes pre-chilled on ice. Frozen aliquots can be stored at -20°C/-80°C for 1 year. Once thawed, aliquots can be kept at 4°C for 7 days maximum. For establishing organoid cultures, we use undiluted Matrigel at a protein concentration between 8.5 and 10.5 mg/mL. Since Matrigel shows batch-tobatch variations due to its animal origin, we recommend first testing the efficiency of one bottle whenever a new batch is purchased, and then using the same batch throughout the entire project, if possible.
Wnt3a-CM is prepared using the mouse L Wnt-3A cell line (ATCC, CRL-2647). Briefly, expand an L Wnt-3A vial in a T75 flask containing DMEM + 10% FBS. Split cells every two days, or when they are 100% confluent. Perform three passages: the first two at a dilution of 1:3, and the third at 1:5.
Collect medium from the third passage, 48 and 96 hours later. Aliquot the CM in 50 mL Falcon tubes, spin at 3000 x rpm for 10 min at 4°C, and filter through a 0.22 µM filter. To assess the quality of the CM, test the batches in the organoid culture over 2-3 passages using the previously produced CM batch, if available, as a reference. Alternatively, the TOP/FOP assay can be used for testing the CM quality. If high-quality CM is produced, it should be divided into aliquots and stored at -20°C for a maximum one year. Once thawed, the aliquots can be stored at 4°C for a maximum of 10 days. The possibility of batch-to-batch variability should always be kept in mind; for this reason we recommend, whenever possible, using the same batch throughout the entire project. Once thawed, aliquots of reagents used for organoid dissociation solution and growth/differentiation media preparation can be stored at 4°C for a maximum of 2 weeks. Avoid repeated freeze-thaw cycles.
ALTERNATIVE PROTOCOL: For isolation of intestinal crypts from a surgical sample, ALTERNATIVE PROTOCOL: For isolation of intestinal crypts from a surgical sample, follow Steps 14-31 follow Steps 14-31 Immediate processing is highly recommended to prevent cell death. If this is not possible, keep the sample at 4°C for a maximum of 5-6 hours before proceeding with the protocol.
2 In this protocol, crypts are embedded in 25 µL-Matrigel drops seeded in a 48-well plate, one drop per well.
Based on the estimated number of crypts of the experiment (as determined in Steps 32-33 Steps 32-33), place the required number of Matrigel aliquots on ice for 1-2 hours before use, pre-warm the required number of 48-well plates in the cell incubator (5% CO 2 , 37°C) about 1-2 hours before use, and prepare the required volume of organoid growth medium.
The volume of the drops can be scaled according to the plate used. For example, 10 µL Matrigel drops can be plated in a well of a 96-well plate, or 4 drops of 25 µL can be plated in a well of a 12-well plate. We cannot guarantee that the integrity of the drop is maintained when higher volumes of Matrigel are used.
3 Wash the biopsies 1-2 times with ≈ 10 mL cold DPBS by gently flipping the tube for a few seconds.
4 Incubate the sample in 10 mL of the antimicrobial cocktail for 15 min at room temperature under gentle agitation on a platform rocker. 5 Wash 1-2 times with ≈ 10 mL DPBS at room temperature by gentle manual flipping for a few seconds to remove the antimicrobial cocktail.
6 Incubate the biopsies with 10 mL of the crypt isolation buffer for 45 min at 4°C under gentle agitation on a platform rocker. 7 Wash once with ≈ 10 mL cold DPBS by gentle manual flipping for a few seconds to remove the crypt isolation buffer. 8 To start collecting the fractions enriched with viable crypts, add 10 mL cold DPBS and shake vigorously by hand for 15-20 seconds. Depending on the sample, isolated crypts released from the tissue can be visible in the suspension in this first wash. Regardless, proceed to the next step.
9 Leave the biopsies to settle at the bottom of the tube and collect about 10 mL of the suspension in a new cold 15 mL Falcon tube using a 5 mL pipette. Add FBS to the suspension to a 5% final concentration to maintain cell viability.
Try to collect as many crypts as possible from each fraction to avoid the possibility that residual crypt are unnecessarily submitted to additional shakes, which could impair their quality.
10 Repeat Steps 8-9 Steps 8-9 until no additional crypts are released. Usually, no crypts are collected after 5-6 shaking cycles. 11 Spin the crypt fractions at 200 x g for 3 min at 4°C. 12 Carefully remove the supernatant from each fraction using a 5 mL pipette and gently resuspend each crypt pellet in 1 mL cold washing medium. Merge the fractions in one 15 mL Falcon tube and spin again at 200 x g for 3 min at 4°C.
13 Resuspend the crypt pellet in cold washing medium. 1-2 mL washing medium are usually enough for an optimal crypt count. At this point, jump to Step 32 Step 32 or Step 33 Step 33 depending if crypt count is desired or not.
Crypts isolated from biopsies of a non-inflamed sigmoid mucosa of a CD patient.
Step 13 includes a Step case.

ALTERNATIVE PROTOCOL ALTERNATIVE PROTOCOL
This protocol is suitable for generating organoids mostly from non-IBD surgical samples , since in surgical samples from IBD patients the excessive inflammation can dramatically limit the isolation of viable crypts. This protocol is similar to the one used for biopsies, with some slight modifications. No extra reagents are required.
Immediate processing is highly recommended to prevent cell death. If this is not possible, keep the sample at 4°C for a maximum of 5-6 hours before proceeding with the protocol.
15 In this protocol, crypts are embedded in 25 µL-Matrigel drops seeded in a 48-well plate, one drop per well.
Based on the estimated number of crypts of the experiment (as determined in Steps 32-33 Steps 32-33), place the required number of Matrigel aliquots on ice for 1-2 hours before use, pre-warm the required number of 48-well plates in the cell incubator (5% CO 2 , 37°C) about 1-2 hours before use, and prepare the required volume of organoid growth medium.
The volume of the drops can be scaled according to the plate used. For example, 10 µL Matrigel drops can be plated in a well of a 96-well plate, or 4 drops of 25 µL can be plated in a well of a 12-well plate. We cannot guarantee that the integrity of the drop is maintained when higher volumes of Matrigel are used.
16 Wash the surgical sample with ≈ 20 mL cold DPBS by gentle manual flipping for a few seconds. Discard the DPBS and repeat the washing until the DPBS becomes clear. 17 Transfer the sample to a 10 cm Petri dish and remove the submucosa and larger contaminating blood vessels with scissors and scalpel. 27 After each shake cycle, leave the sample pieces to settle at the bottom of the tube and collect about 20 mL of the suspension enriched in crypts in a new cold 50 mL Falcon tube using a 10 mL pipette. Add FBS to a 5% final concentration to maintain cell viability.
Try to collect as many crypts as possible from each fraction to avoid the possibility that residual crypt are unnecessarily submitted to additional shakes, which could impair their quality.

Repeat Steps 26-27
Steps 26-27 until the desired number of crypts is obtained.
From a surgical sample large amounts of crypts can be released at each shaking cycle. Stop shaking when it is estimated that the desired number of crypts is obtained (refer to Steps 32-33 Steps 32-33 for crypt counting).
29 Spin the crypt fractions at 150 x g for 3 min at 4°C. 30 Carefully remove the supernatant from each fraction using a 10 mL pipette and gently resuspend each crypt pellet in 1 mL cold washing medium. Merge the fractions in one 50 mL Falcon tube and spin again at 150 x g for 3 min at 4°C. 31 Resuspend the crypt pellet in cold washing medium. Depending on the size of the obtained crypt pellet, between 5-and 10-mL washing medium are usually necessary for an optimal crypt count.
2. Intestinal crypt culture (day 1 to day 3) 32 Place two or three 50 µL-drops of the crypt suspension on a glass slide to check crypt integrity and estimate the average number of crypts in the sample by bright-field microscopy examination.
As a rule, when counting crypts, also include broken ones when the base is present: these will probably give rise to organoids since they still contain a viable stem cell compartment.
Example of a crypt suspension where both intact (circle) and broken (dotted circle) crypts are present. In this example, crypts were isolated from the surgical sample of a non-IBD sigmoid mucosa. 33 Take out the volume of crypts required for the planned experiment and transfer it to a new tube. We suggest embedding 80-100 crypts/25 µL-Matrigel drop seeded in a 48-well plate.
Since the amount of crypts isolated from biopsy samples is limited, Step 32 Step 32 may be skipped when using this type of specimen. Based on our experience, indeed, we calculate that on average 12-16 25µL-Matrigel drops are seeded in a 48-well plate when starting with 6-8 biopsies.
33.1 At this point the isolated crypts can be also used to prepare a short-term crypt culture. In this case we recommend using a lower number of crypts per Matrigel drop (about 50 crypts/25µl-drop). Refer to the following publication as an example of overnight crypt culture: This protocol is suitable for expanding crypt cultures as well as previously This protocol is suitable for expanding crypt cultures as well as previously passaged organoid cultures. passaged organoid cultures.
Observe the crypt/organoid culture under the microscope to define the optimal dilution factor for expansion. Volumes indicated here are adjusted to 48 well-plates.
A passage is usually performed 2-3 days after crypt seeding or every 5-6 days if the organoid culture has been previously expanded. As a rule, the dilution rate is 1:2-1:3 for a crypt culture, and 1:4-1:6 for previously expanded organoid cultures. Either way, dilution should be experimentally adjusted according to the estimated growth rate of the crypt/organoid culture. 39 Thaw the required number of Matrigel aliquots by placing them on ice 1-2 hours before use.
Pre-warm the required number of 48-well plates in a cell incubator (5% CO 2 , 37°C) for about 2-3 hours before use. Prepare organoid growth medium and warm it at room temperature before use. Work with ice-cold tubes and refrigerated centrifuges. Perform all the steps in a sterile biosafety cabinet.

During
Step 42 Step 42, prepare the organoid dissociation solution (without yet adding the dispase to prevent loss of enzymatic activity) and pre-warm it in a water bath at 37°C. Consider that 5 mL solution will be prepared per Falcon tube containing the organoids/Recovery solution mix. 46 Discard the supernatant with a 5 mL pipette first, and then with a P1000 micropipette to completely remove the solution. To resuspend the organoid pellet, first gently add 1 mL of the pre-warmed organoid dissociation solution, pipet gently 2-3 times, and then add the remaining 4 mL. 47 Leave the Falcon tubes in the cell incubator or in a water bath at 37°C for 15-20 min gently inverting the tubes every 5-10 min.
48 Mechanically dissociate the organoids with a G20-G21 needle mounted on a 5 mL syringe. It will take 2-5 min per tube, depending on weather the culture is derived from crypts or from a previously expanded organoid culture. Avoid generating too many bubbles in the solution while syringing.
Organoid dissociation usually takes longer if performed on a previously expanded organoid culture than on a crypt culture. In either case, if big clusters are still present, repeat the syringing until no cell aggregates are visible in the suspension. The extent of organoid dissociation can also be monitored by microscope observation. 49 Spin dissociated organoids at 600-800 x g for 4 min at 4°C. 50 Discard the supernatant with a 5 mL pipette first, and then with a P1000 micropipette to completely remove the solution. 51 Perform 3 additional washes with ≈ 5 mL cold washing medium at 800 x g for 4 min at 4°C to completely remove the dispase. Perform the first two washes in the 15 mL Falcon tube and the last wash in a 1.5 mL Eppendorf tube if the required final volume of Matrigel fits in. At this point single cells from dissociated organoids can be cryopreserved (Step 51.1 Step 51.1) or used for the generation of 2D cultures (Step 51.2 Step 51.2).

For cryopreservation, perform the following:
-Transfer a volume of single cells corresponding to 4-6 original Matrigel drops to a new cold Eppedorf tube.
-Resuspend the cell pellet in 1 mL ice-cold Freezing Medium, transfer it to an ice-cold cryopreservation tube and move the sample to a freezing container.
-Store the sample at -80°C for 24h and transfer it to a liquid nitrogen container for long-term storage.
-To thaw, resuspend the cell-containing cryopreservation tube in 5-10 mL warm washing medium.
-Spin at 700 x g for 4 min at 4°C, resuspend in 1 mL cold washing medium and transfer it to a new cold Eppendorf tube.
-Spin again at 700 x g for 4 min at 4°C.
-At this point, go to Step 52 Step 52.
For cryopreserved cultures, the suggested seeding dilution after thawing is lower than the one used for passaging fresh cultures. We usually work with a dilution ratio of 1:2-1:3. 51.2 For the establishment of a 2D culture, refer for example to the following publication describing in detail how to set a monolayer on conventional culture plates using organoid cultures generated by the proposed protocol: 55 Change the medium with fresh organoid growth medium (without Y-27632) every 48-72h hours. Organoid cultures grown for 5-6 days are typically ready to be used for differentiation (Steps 56-59 Steps 56-59) or for further expansion by repeating the procedure described in this section (Steps 38-55 Steps 38-55).